One of the most intriguing problems in nervous system development is how individual neurons acquire their correct position, morphology and biochemical differentiation. The regularity of the generation of specific neurons in particular locations at particular times during invertebrate embryogenesis suggests that cell lineage patterns determine the phenotype of individual neurons. Many cell fate studies in vertebrates, on the other hand, have concluded that cell lineage plays little if any role in neuronal determination. However, the techniques for lineage analysis in vertebrates are limited and the members of the clones have been characterized by morphology alone; these limitations have not allowed an exhaustive or conclusive study of this issue. Although it is clear in retina that several different phenotypes descend from a single neuroepithelial cell, these studies have not tested: a) the neurotransmitter diversity of the members of a clone; b) the role of cell lineage in the type of neurotransmitter a cell expresses; c) the time at which neurotransmitter phenotypes are determined; or d) the developmental regulation of the numbers of neurons with common neurotransmitter phenotypes. The proposed research will address these questions. The vertebrate retina is an ideal location in which to study neurotransmitter-lineage relationships for many reasons. It contains a diverse array of amacrine cells, in terms of morphology, function and neurotransmitters, and it has been the subject of numerous cell lineage studies in mammals and amphibians. We will supplement these data with information regarding the early mitotic history of each of the different neurotransmitter subtypes of amacrine cells. Also, the presumptive eye fields in the neural plate have been mapped. We will use these maps to study cell lineages and interactions at this important developmental period; this organ and its progenitors are accessible for experimental manipulation throughout development. We propose to: 1. Investigate the Complexity of Xenopus Amacrine Neurotransmitter Phenotypes. Using combinations of antibodies specific for the different neurotransmitters that are known to be synthesized by amacrine cells, we will immunofluorescently double or triple-label tadpole retinae, and characterize the neurotransmitter complexity of this population. 2. Investigate if there is a Relationship Between Different Neurotransmitter Subtypes of Amacrine Cells and the Local Environment in Which they Differentiate by 3-dimensional mapping the different neurotransmitter types of amacrine cells in the retina. 3. Identify the Clonal Origin of the Various Amacrine Neurotransmitter Subtypes by combining lineage tracing techniques with neurotransmitter immunofluorescence. We will characterize the number and spatial location in the retina of each neurotransmitter subtype of amacrine cell which descend from each cleavage stage blastomere. 4. Investigate Whether the Amacrine Cells that Belong to the Same Neuroepithelial Clone Express the Same Neurotransmitters. 5. Investigate the Time at Which Amacrine Neurotransmitter Subtypes are Determined. By ablating a single blastomere or a portion of its clone at sequentially later stages we will identify: a) whether amacrine-producing blastomeres are committed to the amacrine developmental program; b) the time at which amacrine phenotypes are determined; and c) whether compensation (both qualitative and quantitative) by other proliferative cells occurs.